DESCRIPTION: The gene responsible for juvenile neuronal ceroid lipofuscinosis, CLN3, (also denoted CLN3 or Batten's disease) has a homolog in the yeast Saccharonzyces cerevisiae, designated BTNI. Disruption of BTN1 revealed a distinct phenotype, resistance to ANP (or D-(-)-threo-2-amino-1- aboutp-nitrophenyl]-l,3-propanediol). This phenotype is complemented by plasmids containing either the yeast BTN1 gene, or the human CLN3 gene. Thus, the human CLN3 gene functions in yeast. We have established that Btn1p is a vacuolar protein and that btn1-delta strains have an elevated activity of the plasma membrane H+-ATPase in the early phases of growth due to a decreased vacuolar pH. The activity of the plasma membrane H+-ATPase and vacuolar pH return to normal in the later phases of growth, through altered gene expression. Furthermore, we have demonstrated that chloroquine can also return vacuolar pH to normal in btnl-delta strains. Our long term goals are to determine the essential features of CLN3 and Btn1p in yeast, to determine other genes and proteins associated with the BTN1 pathway, and to establish whether yeast can provide a model for phenotypic reversal of Batten disease. Altered vacuolar pH regulation in btnl-delta strains will be investigated with a number of studies, including measurement of the assembly and disassembly of vacuolar H+-ATPaso and analysis of vacuolar content to see if altered pH is due to altered transport of an as yet unidentified substrate. Expression of normal and mutationally altered forms of CLN3 and Btn1p in yeast will be examined to determine the critical regions of the proteins. We propose to identify the proteins interacting with Btnlp, and Btn2p, a novel component of endocytosis with elevated expression in btn]-A strains, which may alter vesicular delivery to the vacuole, using the two hybrid system, as well as by isolating possible complexes using a Btn1p or Btn2p tagged with poly-histidine. Genetic approaches will be utilized for isolating and characterizing genes resembling or interacting with BTN1, including suppressors of btn 1-delta and synthetic lethality/enhancement with b/ni-LI and btn2-delta. A newly uncovered gene, BTN3, and R VS161, are synthetically lethal and synthetically lethal on a non-fermentable carbon source, respectively, in btn1-delta strains and will be studied for physical and genetic interaction with Btn1p. The human homolog to RvsI6lp is amphiphysin, an auto-antigen associated to the neurological disorder, Stiff Man Syndrome. An understanding of the function of Btnlp (and Cln3p) in yeast could furnish valuable information on Batten's disease in humans.